Novel allelic variants in the factor vii gene

ABSTRACT

The presence of at least one of said allelic variants affects the stability and/or functionality of the nucleic acid molecule and/or of the product coded by said nucleic acid molecule. The procedure for analyzing a nucleic acid molecule comprises obtaining said molecule from a biological sample and determining at least one allelic variant from Table 1, said allelic variant affecting the stability and/or functionality of the nucleic acid molecule and/or of the product coded thereby. The isolated product coded by a nucleic acid molecule which includes at least one allelic variant can be used as a medicament.

FIELD OF THE INVENTION

The present invention relates to the field of cardiovascular diseases.

In particular, the present invention relates to the identification ofnew allelic variants in the factor VII gene sequence for determining thepredisposition to a cardiovascular disease.

BACKGROUND OF THE INVENTION

Factor VII is a vitamin K-dependent glycoprotein 15 synthezised in theliver and secreted into the blood as an inactive zymogen at aconcentration of 0.5 μg/ml² (Fair Blood, 1983). Following endothelialdamage, the tissue factor (TF) is exposed and it binds to the FactorVII, setting up a coagulation reaction (Osterud. Proc. Natl. Acad. Sci.USA, 1977; Bauer et al., Blood, 1990).

The gene that encodes the Factor VII is located on 13q34-q.ter (Pfeifferet al., 1982; Gilgenkrantz et al., 1986), contains 9 exons and 8 intronsof 12.8 kb and codes for a protein of 406 amino acids. The complete genesequence for human Factor VII was determined by O'Hara el al. (O'Hara P.J. et al., “Nucleotide sequence of the gene coding for human factor VII,a vitamin K-dependent protein participating in blood coagulation”; Proc.Natl. Acad. Sci. U.S.A. 84:5158-5162 (1987)). The mRNA is polyadenylatedin multiple positions and has an efficient differential splicing. Themature protein has a molecular mass of approximately 50 KDa.

The activated form of the factor VII consists on one heavy chain and onelight chain, both coded by the same gene, and linked by a disulphur bondbetween the cysteine 135 and the cysteine 262 (Hagen et al., 1986). Itcontains two EGF domains (epidermal growth factor domain), one Gladomain (γ-carboxyglutamic acid domain) and one trypsin-like catalyticdomain (Hagen et. al., Natl. Acad. Sci. USA, 1986).

The heavy chain includes the catalytic part of the molecule and theheavy chain contains the Gla domain involved in the Ca²⁺ binding and themembrane binding, which are essential for factor VII activity.

The factor VII heavy-chain variants involve the direct interference inthe activation process or the interruption of the catalytic mechanism,whereas most of the light-chain variants interrupt the interactions withCa²⁺ or with membrane components which results in dysfunctionalmolecules (Zheng et al., Blood Coagul. Fibrinol, 1996).

Heriditary factor VII deficiency is an uncommon disorder showingautosomal recessive inheritance with high penetrance and variableexpressivity (Kupfer et al., 1960; Triplet et al., 1985). It has anincidence of 1 per 500,000 in the general population (Wulf and Hermann.Hum. Mutation 15, 2000) and was recognized for the first time byAlexander et al., 1951. Some of the factor VII gene mutations have beenidentified and affect all domains of the protein, although about 50% ofsaid mutations affect the protease domain (Wulff and Hermann, Hum.mutation, 2000), which indicates that the loss of proteasa function isthe main cause of factor VII deficiency.

In general, the most common forms of disorder involve the presence ofdysfunctional factor VII, which consists in low antigen levels in theplasma and a lengthening of prothrombin time due to defective activityof these molecules.

An absence of factor VII activity in plasma causes severe hemorrhageshortly after birth; indeed, there are studies in which mice deficientin FVII due to targeted disruption of the factor VII gene suffered fatalhemorrhage in the peri-partum period (McVey et al., Hum. mutation,2000).

Moreover, about 30-40% of the variation of FVIIa levels in the generalpopulation can be explained by the existence of polymorphisms in theFVII gene (Bernardi et al., Blood 1996). These polymorphisms or allelicvariants nevertheless show different allelic frequencies in differentpopulations (Green et al., Arterioscler. Thromb., 1991; Bernardi,Marchetti, Pinotti. Arterioscler. Thromb. Vasc. Biol., 1996).

These allelic variants have been associated with varied risk ofsuffering from cardiovascular diseases, although the studies in whichsuch an association has been described are contradictory and in noinstance conclusive (Girelli et al., New England. J. Med., 2000;Iacoviello et al., N. Eng. J. Med., 1988). Furthermore, all the studiessuffer from design errors and lack of statistical power.

The design and methodology used to date to approach the study ofcardiovascular disease were based on investigating for presence of therisk factor in healthy individuals (controls) and disease sufferers(cases) who were unrelated to each other. Where the hypothetical riskfactor was observed more frequently (in statistical terms) in the casesthan in the controls it was concluded that the disease was associatedwith the factor under study. Strictly, however, a relationship ofassociation does not necessary imply causation. This type of study,so-called Association or Case/Control Study, is entirely unsuitable forinvestigating genetic causes of complex illnesses, such ascardiovascular disease (Gambaro et al., Lancet 2000). Conventionalepidemiological studies basically serve to identify environmental causesof illness (such as the smoking habit and lung cancer, oralcontraceptives and venous thrombosis, or a vitamin-C-deficient diet andscurvy), but are highly ineffective when it comes to locating the genesinvolved. However, owing to the widespread use of PCR techniques inclinical laboratories, there are large numbers of Association Studiesthat relate genetic variants (polymorphisms) in certain candidate geneswith all kinds of illnesses. Much confusion has been caused, because theresults relating to a single polymorphism are often contradictory.Neither has the study of cardiovascular disease, for both venous andarterial types, remained free from this methodological perversion norfrom the attendant chaotic collection of results (Girelli et al., NewEng. J. Med., 2000; Iacoviello et al., N. Eng. J. Med. 1998).

DESCRIPTION OF THE INVENTION

The present invention relates to a molecule of nucleic acid comprising asequence of the gene that codes for factor VII, characterized in thatsaid molecule includes at least one allelic variant, said allelicvariant affecting to the stability and/or functionality of said nucleicacid molecule, of the product obtained by transcription of said nucleicacid molecule and/or of the product coded by said nucleic acid molecule.

In the present invention, “nucleic acid molecule” is taken to mean a DNAsequence from the gene coding for factor VII protein. The length of saidsequence is not an essential or restrictive aspect of this invention.

In the present invention, “allelic variant” is taken to mean a geneticvariation in the DNA sequence that codes for factor VII protein, saidgenetic variation involving a pathology, loss or gain of stabilityand/or functionality. In particular, said allelic variant can be adeletion, an insertion or a substitution.

In a first aspect of the invention, therefore, new allelic variants areprovided which have been identified in the gene that codes for factorVII protein.

Surprisingly, the inventors of the present invention have found thatsaid allelic variants affect not only the functionality of the factorVII, but also the levels at which that protein is found. This is due tothe fact that said variants can affect not only the nucleic acidmolecule (DNA) but also the one transcribed from said molecule (RNA) andthe protein. For example, if the allelic variant gives rise to anincrease of the RNA stability, higher plasma levels of factor VIIprotein will be obtained; if the allelic variant affects an exon (thatis, a protein-coding region) the functionality of the factor VII will beaffected; if the allelic variant is on an intron (that is, on anon-coding region) DNA and/or RNA stability can be affected, alteringthe blood levels of FVII (increasing or decreasing said levels).

In this invention, “said variant affecting stability and/orfunctionality” is taken to mean an allelic variant that leads to anincrease or a reduction of the stability, whether of DNA or RNA, and/orto increase in or loss of FVII function.

The human gene sequence that codes for FVII is known in the art(sequence published by O'Hara, P. J.; Grant, F. J.; Haldeman, B. A.;Grey, C. L.; Insley, M. Y.; Hagen, F. S.; Murray, M. J.: “Nucleotidesequence of the gene coding for human factor VII, a vitamin K-dependentprotein participating in blood coagulation”. Proc. Net. Acad. Sci. 84:5158-5162, 1987. Access number in PubMed ID:30375737).

The allelic variants identified in the present invention are located onthe promoter region of intron 1, intron 2, exon 3, intron 3, intron 5,exon 6, intron 7, intron 8, exon 9, in the 3′-UTR region (region 3′untranslatable to protein, but contains regulating sequences), as shownin the following table: TABLE 1 allelic variants identified in thepresent invention. SNP (Single Nucleotide Polymorphism) variation of abase (nucleotide) in the DNA sequence. Nucleotide Allelic O'Hara et al.Variant Position Type −3216 C/T Promoter SNP −2987 C/A Promoter SNP −668A/C Promoter SNP −628 A/G Promoter SNP −402 G/A Promoter SNP −401 G/TPromoter SNP −323 Ins 0/10 Promoter Insertion −122 T/C Promoter SNP 73G/A Intron 1 SNP 260 A/G Intron 1 SNP 364 G/A Intron 1 SNP 698 T/CIntron 1 SNP 705 G/A Intron 1 SNP 710 C/G Intron 1 SNP 723 IVS1 Intron 1VNTR 799 T/C Intron 1 SNP 806 G/A Intron 1 SNP 811 C/G Intron 1 SNP 833T/C Intron 1 SNP 3.171 G/A Intron 2 SNP 3.294 G/A Intron 2 SNP 3.380 C/TIntron 2 SNP 3.423 G/T Intron 2 SNP 3.928 Q35Q G/A Exon 3 SNP 4.003 G/AIntron 3 SNP 5.191 A/G Intron 3 SNP 5.503 T/A Intron 3 SNP 6.331 G/AIntron 5 SNP 6.448 G/T Intron 5 SNP 6.452 G/T Intron 5 SNP 6.461 IVS5Intron 5 VNTR 7.161 G/C Intron 5 SNP 7.453 T/G Intron 5 SNP 7.729 G/AIntron 5 SNP 7.880 H115H C/T Exon 6 SNP 8.695 G/A Intron 6 SNP 9.724IVS7 Intron 8 VNTR 9.734 A/G Intron 8 SNP 9.779 T/C Intron 8 SNP 9.792G/A Intron 8 SNP 9.847 C/T Intron 8 SNP 10.524 G/A Intron 8 SNP 10.534T/C Intron 8 SNP 10.799 A294V C/T Exon 9 SNP 10.914 S333S G/A Exon 9 SNP10976 R353Q G/A Exon 9 SNP 11.293 Ins AA 3′-UTR Insertion 11.622 Del AG3′-UTR SNP 11.912 G/A 3′-UTR SNP

The numbering of the allelic variants described in the table is based onthe numbering of the sequence of the human factor VII coding genepublished by O'Hara.

The first column indicates the position in which an allelic variant wasdetected, taking O'Hara's published numbering sequence as a reference.The upper-case letters (in the second column) show which is the allelicvariant in a specific position. For example, −3216 C/T means that inposition −3216 (which is in the region of the promoter) the normalallele is a C (cytosine) and the allelic variant is T (thymidine); 11293Ins AA means that two adenine nucleotides are inserted at position11293; 11622 of the AG means that there is deletion of two nucleotides,adenine and guanine, at position 11622.

In a second aspect, the inventors of the present invention have foundthat identification of said allelic variants in a nucleic acid moleculeindicates that the patient might develop a cardiovascular disease, dueto the fact that said allelic variants may be functional (identified onthe nucleic acid molecule's exons), affecting the total or partialfunction of the protein coded by said molecule and, therefore, having aneffect on the coagulation process in which said protein is involved.

Indeed, the protein (factor VII) coded by a nucleic acid molecule inaccordance with the present invention can have altered its stability,its secretion from the cell to the plasma, the average life in theplasma, etc.

The nucleic acid molecule can itself be altered by the presence of atleast one of the allelic variants of Table 1, in terms of transcriptionrate, average life of messenger RNA, rate of translation to protein inthe ribosomes, etc.

Due to all this, this invention relates to proteins coded by a nucleicacid molecule that includes at least one allelic variant of Table 1, foruse as a medicament.

For example, if the allelic variant is located on an intron of saidnucleic acid molecule, a more stable FVII protein that remains for agreater time in the plasma can be obtained and used for administrationto patients with coagulation problems.

Another aspect of this invention is a procedure for analyzing a nucleicacid molecule, characterized in that it comprises obtaining saidmolecule from a biological sample and determining at least one allelicvariant of Table 1, said allelic variable affecting the stability and/orfunctionality of said nucleic acid molecule and/or of the product codedthereby.

Under yet another aspect, this invention relates to a device fordetermining a predisposition to a cardiovascular disease which includesat least one of the oligonucleotides identified in Table 3 (see below).

Advantageously, if the patient's DNA sample is hybridized with at leastone of the oligonucleotides identified in Table 3, this will indicatethat it has at least one allelic variant in the factor VII gene and,therefore, the origin of the altered function of the factor VII can bedetermined.

A specific treatment can thus be designed for preventing acardiovascular disease in patients who have not yet developed one butwho present at least one allelic variant; a treatment can be designedthat is specifically for palliating the factor VII dysfunction; or theproduct encoded by said nucleic acid molecule can be used to treat anillness associated with the coagulation cascade.

This invention therefore provides new allelic variants identified on thegene that codes for factor VII and affect the stability and/orfunctionality of said nucleic acid molecule and/or of the product codedthereby.

Advantageously, detection of said allelic variants not only permitsdetection of a predisposition to a cardiovascular disease(thrombosis-associated) but also means that the proteins that are codedby the nucleic acid molecules which include at least one of the allelicvariants of Table 1 can be used as a medicament for the treatment ofcomplications associated with thrombosis or with coagulation.

The following example is included by way of non-restrictive illustrativeexample.

EXAMPLES Example 1 Procedure for Identifying the Allelic Variants of thePresent Invention

1. Blood Extraction

The DNA was extracted from white blood cells (leukocytes) fromnon-related persons with plasma FVII levels very much higher or lowerthan those which an expert in the art would consider to be normal for anaverage population. The blood samples were taken from the anticubitalvein and anticoagulated immediately with 1/10 volume of sodium citrate0.129 M.

The platelet-deficient plasma was obtained by centrifuging at 2000 g for20 minutes, following which it was frozen and kept at −40° C. until thetime of analysis.

2. Isolation and Amplification of the DNA.

The DNA was purified from the nuclei of leukocytes using the proceduredescribed by Miller et al. (Miller et al., Nucl Ac Res 16 (3): 1215,1988).

The FVII gene was analysed in various overlapping fragments that coveredthe entire gene sequence. The technique used for this analysis wasPolymerase Chain Reaction (PCR). The primers used to amplify thesefragments are shown in Tables 2 and 3. TABLE 2 primers used to amplifythe fragments obtained by PCR Amplification primers Other internalprimers Fragment 1.1 71-72 Fragment 1.2 71.4-72   Fragment 2 73-7473.1/73.2/74.1/74.3 Fragment 3 75-76 Fragment 4 77-78 77.1/78.1 Fragment5.1  79B-710B Fragment 5.2  79C-710.1 710.1/710G Fragment 6.1711.2-712.1 711.5 Fragment 6.2 711.1-712.2 Fragment 6.3 711.2-712  711.4 Fragment 7 713-714 714.1/714.2/713.2/713.3 Fragment 8 715-716715.1/716.1

The various fragments of the FVII gene were numbered consecutivelyaccording to the order of analysis.

The primers were also numbered consecutively, the even numberspertaining to forward primers and the odd numbers to reverse primers.Those skilled in the art know that in order to amplify a DNA fragment byPCR a forward primer and another reverse primer (complementing the DNAchain to be amplified) are always needed.

Included schematically are the sequences of the primers used (sequencesNO:1 to NO:36). TABLE 3 Primer SEQ. NO. F715.1* 1 F72 2 F73 3 F74 4 F775 F78 6 F712 7 F713 8 F714 9 F715 10 F716 11 F711.2 12 F712.1 13 F711.114 F712.2 15 F710.1 16 F711.3 17 F716.1* 18 F714.1* 19 F73.1* 20 F77.1*21 F78.1* 22 F713.2* 23 F73.2* 24 F713.3* 25 F714.2* 26 F71.4* 27F711.5* 28 F711.4* 29 F79A 30 F710A 31 F79B 32 F710B 33 F79C 34 F710C 35F710G 36

The methodology followed for the PCR was standard in the art. Briefly,each fragment was amplified using GeneAmp PCR System 9700 (PE AppliedSystems). The PCR products were generated in 50 μl of reaction mixturescontaining 200 ng of genomic DNA, 0.5 U of Taq DNA polymerase (BiotaqDNA polymerase. Bioline), and the primers indicated in Tables 2 and 3,at a concentration of 0.5 μM each, the dNTPs at a concentration of 0.05mM each, 1.5 mM MgCl₂ (1 mM MgCl₂ for fragment 1), and 5% DMSO (no DMSOin fragment 1 in the buffer for the PCR 1× Bioline).

The PCR programme was started with 5 min. at 94° C. during the initialdenaturation, followed by 30 cycles of amplification that consisted in 1min. at 94° C., 1 min. at hybridisation temperature (57° C. forfragments 1, 2, 7, 9 and 11, 59° C. for fragments 3 and 8, and 61° C.for fragment 10, of Table 1) and 2 min. at 72° C. The extension time wasincreased to 10 min. in the last cycle.

The amplified fragments were submitted to electrophoresis on an agarosegel at 1% for the control.

3. Sequencing of the DNA.

An enzymatic sequencing or dideoxynucleotides method was used, whoseprinciple is based on the synthesis of a DNA chain by using a DNApolymerase based on a previously denatured DNA mould (fragment to besequenced). In this case the PCR technique (mentioned above) was used,with four types of bases comprising the DNA in the form ofdideoxynucleotides (ddNTPs), each marked with a different fluorescence.

Said fluorescence marker can be any of those known to those skilled inthe art, such as the commercially available BigDyes (Apply-Biosystems).

It is at this step that synthesis of the DNA was interrupted by addingone of the ddNTPs. Thus a large number of fragments of different sizeswere obtained and separated using continuous capillary electrophoresis.Each one of these fragments incorporates a fluorescent ddNTP thatcorresponds to a base making up the DNA chain. The colour of eachfragment is determined when the fluorochrome (the ddNTP's fluorescentmaterial) is excited by a laser, thereby producing a signal which isreceived by a photomultiplier and transmitted to a computer.

Computer analysis of the signals allows the sequence of the studiedfragment to be determined. This technique is carried out using anautomatic DNA sequencer (in our case, an ABI-310 model from ApplyBiosystems).

All the allelic variants were identified by direct sequencing of theFVII gene.

The PCR-amplified fragments were purified, and the dNTPs and theunincorporated oligonucleotides removed using Quiagen QIAquick PCRPurification Kit columns before being sequenced.

The sequencing reaction was carried out in a volume of 10 μl, containing3 μl of the purified DNA fragment, 4 μl of DNA Sequencing Kit BigDyeTerminator Cycle Sequencing Ready Reaction (Applied Biosystems), 5% ofdimethylsulfoxide (DMSO vol/vol) and 0.32 μM of the oligonucleotide forsequencing (Table 3).

The sequencing program comprised an initial step at 3′ at 94° C.,following by 25 cycles with the following routine: 10 seconds at 96° C.,5 seconds of hybridisation at 50° C. and 4 minutes at 60° C. Thesequences were carried out in an ABI PRISM 310 Genetic Analyzer.

Accordingly, the allelic variants of Table 1 were identified.

1. Molecule of nucleic acid which comprises a sequence of the gene thatcodes for factor VII, wherein said molecule includes at least oneallelic variant, said allelic varient being one of those identified inTable 1: Nucleotide Allelic O'Hara et al. Variant Position Type −3216C/T Promoter SNP −2987 C/A Promoter SNP −668 A/C Promoter SNP −628 A/GPromoter SNP 73 G/A Intron 1 SNP 260 A/G Intron 1 SNP 364 G/A Intron 1SNP 698 T/C Intron 1 SNP 705 G/A Intron 1 SNP 710 C/G Intron 1 SNP 723IVS1 Intron 1 VNTR 799 T/C Intron 1 SNP 806 G/A Intron 1 SNP 811 C/GIntron 1 SNP 833 T/C Intron 1 SNP 3.171 G/A Intron 2 SNP 3.294 G/AIntron 2 SNP 3.380 C/T Intron 2 SNP 3.423 G/T Intron 2 SNP 3.928 Q35QG/A Exon 3 SNP 4.003 G/A Intron 3 SNP 5.191 A/G Intron 3 SNP 5.503 T/AIntron 3 SNP 6.331 G/A Intron 5 SNP 6.448 G/T Intron 5 SNP 6.452 G/TIntron 5 SNP 6.461 IVS5 Intron 5 VNTR 7.161 G/C Intron 5 SNP 7.453 T/GIntron 5 SNP 7.729 G/A Intron 5 SNP 7.880 H115H C/T Exon 6 SNP 8.695 G/AIntron 6 SNP 9.724 IVS7 Intron 8 VNTR 9.734 A/G Intron 8 SNP 9.779 T/CIntron 8 SNP 9.792 G/A Intron 8 SNP 9.847 C/T Intron 8 SNP 10.524 G/AIntron 8 SNP 10.534 T/C Intron 8 SNP 10.799 A294V C/T Exon 9 SNP 10.914S333S G/A Exon 9 SNP 10976 R353Q G/A Exon 9 SNP 11.293 Ins AA 3′-UTRInsertion 11.622 Del AG 3′-UTR SNP 11.912 G/A 3′-UTR SNP


2. Isolated product coded by a nucleic acid molecule according to claim1 for use as a medicament.
 3. Allele-specific oligonucleotide whichhybridizes with a nucleic acid molecule as claimed in claim 1, in whichthe nucleotide of the polymorphic locus of said allele-specificoligonucleotide is different from the nucleotide of the polymorphiclocus of the reference allele.
 4. Oligonucleotide as claimed in claim 3,wherein it is a probe.
 5. Oligonucleotide as claimed in claim 3, whereinit is one of the group consisting in SEQ ID N^(o): 1 to
 36. 6. Procedurefor analysis of a nucleic acid molecule, wherein it comprises obtainingsaid molecule from biological sample and determining at least oneallelic variant from Table 1, said allelic variant affecting thestability and/or functionality of the nucleic acid molecule and/or ofthe product coded thereby.
 7. Diagnostic device for determining apredisposition to a cardiovascular disease, wherein it includes anoligonucleotide; said oligonucleotide is different from the nucleotideof the polymorphic locus of the reference allele.
 8. Use of a moleculeof nucleic acid according to claim 1 for the development of therapeutic,preventive or diagnostic approaches for the treatment of acardiovascular disease.
 9. Use of an isolated product according to claim2 for the manufacture of a medicament for the treatment of acardiovascular disease.